Localizing Proton-Mediated Inhibitory Feedback at the Retinal Horizontal Cell-Cone Synapse with Genetically-Encoded pH Probes.

Lateral inhibition in the vertebrate retina depends on a negative feedback synapse between horizontal cells (HCs) and rod and cone photoreceptors. A change in pH is thought to be the signal for negative feedback, but its spatial profile in the synaptic cleft is unknown. Here we use three different membrane proteins, each fused to the same genetically-encoded pH-sensitive Green Fluorescent Protein (GFP) (pHluorin), to probe synaptic pH in retina from transgenic zebrafish (Danio rerio) of either sex. We used the cone transducin promoter to express SynaptopHluorin (pHluorin on vesicle-associated membrane protein (VAMP2)) or CalipHluorin (pHluorin on an L-type Ca2+ channel) and the HC-specific connexin-55.5 promoter to express AMPApHluorin (pHluorin on an AMPA receptor). Stimulus light led to increased fluorescence of all three probes, consistent with alkalinization of the synaptic cleft. The receptive field size, sensitivity to surround illumination, and response to activation of an alien receptor expressed exclusively in HCs, are consistent with lateral inhibition as the trigger for alkalinization. However, SynaptopHluorin and AMPApHluorin, which are displaced farther from cone synaptic ribbons than CalipHluorin, reported a smaller pH change. Hence, unlike feedforward glutamatergic transmission, which spills over to allow cross talk between terminals in the cone network, the pH change underlying HC feedback is compartmentalized to individual synaptic invaginations within a cone terminal, consistent with private line communication.SIGNIFICANCE STATEMENT Lateral inhibition (LI) is a fundamental feature of information processing in sensory systems, enhancing contrast sensitivity and enabling edge discrimination. Horizontal cells (HCs) are the first cellular substrate of LI in the vertebrate retina, but the synaptic mechanisms underlying LI are not completely understood, despite decades of study. This paper makes a significant contribution to our understanding of LI, by showing that each HC-cone synapse is a "private-line" that operates independently from other HC-cone connections. Using transgenic zebrafish expressing pHluorin, a pH-sensitive GFP variant spliced onto three different protein platforms expressed either in cones or HCs we show that the feedback pH signal is constrained to individual cone terminals, and more stringently, to individual synaptic contact sites within each terminal.

Feline Neovascular Vitreoretinopathy and Anterior Segment Dysgenesis With Concurrent Glaucoma in Domestic Cats.

Feline neovascular vitreoretinopathy (FNV) is a newly recognized rare condition affecting kittens and young domestic cats. This study investigated the clinical and pathologic findings in 22 cats with FNV. In affected cats, ophthalmoscopy of the fundus (when visible) revealed avascular peripheral retinae and epiretinal vascular membranes. Frequent nonspecific clinical findings were buphthalmos ( n = 21), medically uncontrollable glaucoma ( n = 22), and lenticular abnormalities ( n = 13). Anterior segment dysgenesis (ASD) was detected clinically in affected cats ( n = 6). The fellow eye was affected in 11 of 18 cats to a variable degree or appeared clinically normal in 7 of 18 cats. The globes were examined histologically and using immunohistochemistry for vimentin, glial fibrillary acidic protein (GFAP), synaptophysin, neurofilament, laminin, factor VIII-related antigen (FVIII-RA), and smooth muscle actin (SMA). Histologically, diagnostic features included laminin-positive epiretinal vascular membranes affecting the central retina, with an avascular peripheral retina and gliosis. Enucleated globes exhibited multiple additional abnormalities, including corneal disease ( n = 15), anterior segment dysgenesis ( n = 21), lymphoplasmacytic anterior uveitis ( n = 19), peripheral anterior synechiae ( n = 20), retinal degeneration ( n = 22), and retinal detachment ( n = 19). Gliotic retinae labeled strongly for GFAP and vimentin with reduced expression of synaptophysin and neurofilament, consistent with degeneration or lack of differentiation. While an avascular peripheral retina and epiretinal fibrovascular membranes are also salient features of retinopathy of prematurity, there is no evidence to support hyperoxic damage in cats with FNV. The cause remains unknown.

Protozoal infections of the cornea and conjunctiva in dogs associated with chronic ocular surface disease and topical immunosuppression.

OBJECTIVE: To describe five cases of protozoal keratitis or conjunctivitis in dogs with chronic preexisting ocular surface disease treated with long-term immunosuppression. ANIMALS STUDIED: Five dogs that developed corneal or conjunctival mass lesions. PROCEDURES: The database of the Comparative Ocular Pathology Laboratory of Wisconsin was searched for canine cases diagnosed with corneal or conjunctival protozoal infection. Five cases were identified, and tissues were examined using routine and special histochemical stains: immunohistochemical labels for Neospora caninum, Toxoplasma gondii, and Leishmania spp., and tissue sample PCR for Leishmania spp., Trypanosoma cruzi, tissue coccidia (i.e., T. gondii/Sarcocystis/Neospora), piroplasms, trichomonads, and Acanthamoeba. Electron microscopy was performed for two cases, and serology for N. caninum and T. gondii was available for three cases. RESULTS: Preexisting ocular diseases included keratoconjunctivitis sicca and pigmentary keratitis (n = 4) and pyogranulomatous meibomian adenitis (n = 1). All dogs were treated with tacrolimus or cyclosporine for at least 1.2 years. Dogs were presented with fleshy corneal or conjunctival masses that were clinically suspected to be neoplastic (n = 4) or immune mediated (n = 1). Histologic examination revealed granulomatous inflammation with intralesional protozoal organisms. Amoeba (n = 2), T. gondii (n = 2), or Leishmania mexicana (n = 1) were identified using molecular techniques. Serological tests were negative. CONCLUSIONS: Protozoal keratitis and conjunctivitis without systemic involvement appears rare and may be associated with chronic preexisting ocular surface disease treated with long-term immunosuppression. Based upon clinical appearance, lesions could be confused with neoplasia. This is the first report of amoebic keratoconjunctivitis in dogs and of L. mexicana in dogs in the United States.

Differences in ocular parameters between diurnal and nocturnal raptors.

OBJECTIVE: To establish and compare normal ocular parameters between and within diurnal and nocturnal raptor groups. ANIMALS STUDIED: Eighty-eight ophthalmically normal raptors of six nocturnal and 11 diurnal species were studied. PROCEDURE: Tear production was measured using Schirmer tear test (STT) and phenol red thread test (PRTT), and applanation tonometry was conducted. Ultrasonographic measurements of axial length (AL), mediolateral axis (ML), vitreous body (VB), and pecten length (PL) were recorded, and conjunctival cultures were obtained. RESULTS: A weak correlation (R = 0.312, P = 0.006) was found between PRTT and STT. Tear production was significantly lower in nocturnal species (P < 0.001), but no difference was observed in intraocular pressure (IOP). VB and PL were significantly longer in diurnals (P < 0.001 and P = 0.021, respectively), and no significant difference was observed in AL and ML. When comparing results within these groups, there was a significant difference between most species for all parameters except IOP. Fifty-one percent of the examined raptors were positive for mycology or bacteriology, either on culture or PCR. The most common infectious agent isolated was Staphylococcus spp. CONCLUSIONS: Phenol red thread test and STT are both valid methods to measure tear production; however, a separate baseline must be determined for each species using these methods, as the results of one method cannot be extrapolated to the other. Due to significant differences observed within diurnal and nocturnal species, it appears that a more intricate division should be used when comparing these parameters for raptors, and the classification of diurnal or nocturnal holds little significance in the baseline of these data.

Manifestations of systemic disease in the retina and fundus of cats and dogs.

The fundus is unique in that it is the only part of the body that allows for a noninvasive and uninterrupted view of vasculature and nervous tissue. Utilization of this can be a powerful tool in uncovering salient incidental findings which point to underlying systemic diseases, and for monitoring response to therapy. Retinal venules and arterioles allow the clinician to assess changes in vascular color, diameter, outline, and tortuosity. The retina and optic nerve may exhibit changes associated with increased or decreased thickness, inflammatory infiltrates, hemorrhages, and detachments. While some retinal manifestations of systemic disease may be nonspecific, others are pathognomonic, and may be the presenting sign for a systemic illness. The examination of the fundus is an essential part of the comprehensive physical examination. Systemic diseases which may present with retinal abnormalities include a variety of disease classifications, as represented by the DAMNIT-V acronym, for Degenerative/Developmental, Anomalous, Metabolic, Neoplastic, Nutritional, Inflammatory (Infectious/Immune-mediated/ischemic), Toxic, Traumatic and Vascular. This review details systemic illnesses or syndromes that have been reported to manifest in the fundus of companion animals and discusses key aspects in differentiating their underlying cause. Normal variations in retinal anatomy and morphology are also considered.

Controlling Horizontal Cell-Mediated Lateral Inhibition in Transgenic Zebrafish Retina with Chemogenetic Tools.

Horizontal cells (HCs) form reciprocal synapses with rod and cone photoreceptors, an arrangement that underlies lateral inhibition in the retina. HCs send negative and positive feedback signals to photoreceptors, but how HCs initiate these signals remains unclear. Unfortunately, because HCs have no unique neurotransmitter receptors, there are no pharmacological treatments for perturbing membrane potential specifically in HCs. Here we use transgenic zebrafish whose HCs express alien receptors, enabling cell-type-specific control by cognate alien agonists. To depolarize HCs, we used the Phe-Met-Arg-Phe-amide (FMRFamide)-gated Na+ channel (FaNaC) activated by the invertebrate neuropeptide FMRFamide. To hyperpolarize HCs we used a pharmacologically selective actuator module (PSAM)-glycine receptor (GlyR), an engineered Cl- selective channel activated by a synthetic agonist. Expression of FaNaC or PSAM-GlyR was restricted to HCs with the cell-type selective promoter for connexin-55.5. We assessed HC-feedback control of photoreceptor synapses in three ways. First, we measured presynaptic exocytosis from photoreceptor terminals using the fluorescent dye FM1-43. Second, we measured the electroretinogram (ERG) b-wave, a signal generated by postsynaptic responses. Third, we used Ca2+ imaging in retinal ganglion cells (RGCs) expressing the Ca2+ indicator GCaMP6. Addition of FMRFamide significantly decreased FM1-43 destaining in darkness, whereas the addition of PSAM-GlyR significantly increased it. However, both agonists decreased the light-elicited ERG b-wave and eliminated surround inhibition of the Ca2+ response of RGCs. Taken together, our findings show that chemogenetic tools can selectively manipulate negative feedback from HCs, providing a platform for understanding its mechanism and helping to elucidate its functional roles in visual information processing at a succession of downstream stages.

Retinoic Acid Induces Hyperactivity, and Blocking Its Receptor Unmasks Light Responses and Augments Vision in Retinal Degeneration.

Light responses are initiated in photoreceptors, processed by interneurons, and synaptically transmitted to retinal ganglion cells (RGCs), which send information to the brain. Retinitis pigmentosa (RP) is a blinding disease caused by photoreceptor degeneration, depriving downstream neurons of light-sensitive input. Photoreceptor degeneration also triggers hyperactive firing of RGCs, obscuring light responses initiated by surviving photoreceptors. Here we show that retinoic acid (RA), signaling through its receptor (RAR), is the trigger for hyperactivity. A genetically encoded reporter shows elevated RAR signaling in degenerated retinas from murine RP models. Enhancing RAR signaling in healthy retinas mimics the pathophysiology of degenerating retinas. Drug inhibition of RAR reduces hyperactivity in degenerating retinas and unmasks light responses in RGCs. Gene therapy inhibition of RAR increases innate and learned light-elicited behaviors in vision-impaired mice. Identification of RAR as the trigger for hyperactivity presents a degeneration-dependent therapeutic target for enhancing low vision in RP and other blinding disorders.

Outcome of iridociliary epithelial tumour biopsies in dogs: a retrospective study.

To evaluate the outcome of eyes with a confirmed iridociliary epithelial tumour (ICET) following biopsy. Forty-two specimens were selected from the Comparative Ocular Pathology Laboratory of Wisconsin database, including 11 globes enucleated following ICET biopsy and 31 iridociliary biopsies with a confirmed ICET. Histopathology was performed for all specimens. When identified, the corneal surgical wound was examined in enucleated globes. Tumour type and margins were determined for biopsy specimens and follow-up was obtained when possible. Biopsies were performed for diagnosis, debulking or excision. 30/31 biopsies had dirty margins, and iridociliary adenomas were indistinguishable from adenocarcinomas by biopsy. Upon biopsy submission 5/23 biopsies were reported as incisional and 18/23 as excisional. Follow-up information was obtained for 14/18 of those reported as excisional. 8/14 had documented recurrence within 5.0±5.6 months and 6/14 had no recurrence at 21.5±13.6 months postoperatively. Three enucleated globes were diagnosed with iridociliary adenocarcinomas and eight with iridociliary adenomas. The corneal surgical wound was sampled in 8/11 globes. There was a synechia to the surgical wound in 3/8 globes, and in 3/8 globes there were neoplastic cells within or adjacent to the surgical wound. The postoperative success of ICET excision is highly variable; complete excision is rarely achieved and recurrence is common. Biopsy effects on ocular tissues may result in synechia and other surgical complications. ICET can be diagnosed by biopsy, but adenomas are indistinguishable from adenocarcinomas.

Keratitis in six dogs after topical treatment with carbonic anhydrase inhibitors for glaucoma.

CASE DESCRIPTION: 6 dogs (10 eyes) with keratitis following long-term topical treatment with a carbonic anhydrase inhibitor (CAI) were evaluated. In 4 dogs (6 eyes), CAI treatment was discontinued. Three dogs (4 eyes) underwent enucleation because of end-stage corneal disease. One dog was treated differently in each eye and thus was represented in both aforementioned groups. CLINICAL FINDINGS: Following initiation of treatment with a CAI (ie, brinzolamide or dorzolamide), the median time to development of severe ocular signs was 266 days (range, 133 to 679 days). Clinically severe ocular signs included ulcerative and nonulcerative perilimbal keratitis or severe diffuse keratitis with marked vascularization. The keratitis was refractory to treatment with anti-inflammatory medications. Histologic and immunohistochemical examination of enucleated globes was performed in 3 affected dogs and in 1 dog with keratitis that recovered. Corneal lesions included 2 distinct inflammatory infiltrates with plasma cells predominating in the anterior stroma and both T cells and neutrophils in the epithelium. Stromal plasma cells and overlying epithelium exhibited strong positive immunoreactivity for IgG. TREATMENT AND OUTCOME: Topical CAI treatment was discontinued in 4 dogs after a median of 209 days (range, 44 to 433 days), and in these dogs, clinical improvement was evident within 2 to 4 days of CAI treatment cessation. Signs of keratitis resolved in 12 to 25 days in these 4 dogs, and median follow-up time after CAI discontinuation was 25.5 months (range, 6 to 42 months), during which time signs of corneal disease did not recur. CLINICAL RELEVANCE: On the basis of this small series, presumed topical CAI-associated keratitis in dogs appeared to be an uncommon immune-mediated disease that was not responsive to corticosteroid treatment. Affected patients improved rapidly, but only after discontinuation of CAI treatment. In dogs with glaucoma, clinicians should consider the development of punctate keratopathy and severe diffuse keratitis as potential adverse effects related to topical administration of CAIs, even after previously uneventful long-term use.

Presumed primary intraocular chondrosarcoma in cats.

Following unilateral enucleation, 4 Domestic Shorthair cats with an average age of 12.5 years (range: 9-16 years) were histologically diagnosed with a presumed primary intraocular chondrosarcoma at the Comparative Ocular Pathology Laboratory of Wisconsin (Madison, Wisconsin). Medical records and follow-up were available for 3 of the 4 cats. Clinically, only 1 eye was affected in each cat; a mass lesion was noted in 2 cats, and a neoplasm was suspected in the other 2 cats. Grossly, 3 tumors presented as coalescing, poorly demarcated, white, friable masses filling the vitreous and intraocular chambers; 1 tumor presented as a solitary, well-demarcated, tan mass involving the iris and ciliary body. Histologically, all 4 neoplasms were composed of haphazardly arranged plump neoplastic spindle cells surrounded by irregular islands and thick trabeculae of abundant, variably basophilic, and Alcian blue-positive chondromatous matrix. None of the cats presented histologically or clinically with signs suggestive of feline posttraumatic ocular sarcoma. Two cats are still alive and healthy 6 months and 3 years following enucleation. One cat died 6 months following enucleation; however, this cat suffered from poorly controlled diabetes mellitus, and the cause of death is undetermined. No other tumors or skeletal lesions were identified that could suggest a metastatic tumor to the eye. The origin of primary intraocular chondrosarcoma is unclear, but is presumed to be ocular multipotent mesenchymal stem cells. Four cases of intraocular chondrosarcoma in cats not associated with the posttraumatic sarcoma complex of intraocular tumors are described.

Comparison of tear pH in dogs, horses, and cattle.

OBJECTIVE: To determine baseline tear pH in dogs, horses, and cattle by use of a microelectrode. ANIMALS: 28 dogs, 24 horses, and 29 cattle. PROCEDURES: Under manual restraint, tears were collected from each subject's left eye with cotton spears. A Schirmer tear test was performed in the right eye. Tears were extracted from the spears by centrifugation. Tear volume was measured, pH was determined with a microelectrode, and total solids (TS) concentration was measured by refractometry. RESULTS: Mean ± SD pH of tears in cattle, dogs, and horses was 8.32 ± 0.14, 8.05 ± 0.26, and 7.84 ± 0.30, respectively. Tear pH was significantly higher in cattle versus dogs and horses and in dogs versus horses. Mean ± SD TS concentration in horses, cattle, and dogs was 2.04 ± 1.29 g/dL, 1.07 ± 0.60 g/dL, and 0.33 ± 0.18 g/dL, respectively. Total solids concentration was significantly higher in horses versus cattle and dogs and in cattle versus dogs. Schirmer tear test results for all animals were within the species reference range. CONCLUSIONS AND CLINICAL RELEVANCE: Tear pH in all 3 species differed from that of published blood pH values and the pH of common topically administered ophthalmic medications. These fndings may have implications for variations in ocular flora and defense mechanisms, susceptibility to ocular disease, and success or comfort of topical treatment.